Context:
Conservation of vulnerable and endangered species requires a comprehensive understanding of their distribution and habitat requirements, so as to implement effective management strategies. Visual scat surveys are a common non-invasive method for monitoring populations. However, morphological similarity of scats among sympatric species presents a problem for accurate identification. Visual misidentifications of scats can have major impacts on the accuracy of abundance and distribution surveys of target species, wasting resources and misdirecting management and conservation actions. DNA identification of scats can overcome this issue, while simultaneously providing a rich source of genetic information for population and dietary studies.

Aims:
We developed a simple and reliable method to identify morphologically similar macropod scats from eight sympatric species in north-eastern Australia, using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of a portion of the mtDNA ND2 gene.

Methods:
We identified a short (275-bp) polymorphic region of ND2, which is easily amplifiable from degraded DNA, developed a primer set, and identified a set of three restriction endonucleases (AluI, BstNI and HphI) which, in combination, can discriminate among the eight target species. So as to test the effectiveness of this protocol, we collected 914 macropod scats from 53 sites in the north-eastern Australia.

Key results:
In total, 406 of these scats were extracted, with 398 (98%) containing amplifiable macropod DNA. All 398 scats were subsequently identified to species by using our RFLP protocol. Sequencing of a subset of these samples confirmed the accuracy of the test. Species identification of scats by using DNA identified eight species of macropods, five of which were outside their documented distributions, one of which was ~400 km.

Conclusions:
Our PCR–RFLP method is a simple and efficient means to identify macropod scats to species, eliminating the need for sequencing, which is costly, time-consuming and requires additional laboratory equipment.

Implications:
The method allows for rapid and non-invasive assessment of macropod species and is particularly useful for surveying populations across multiple sites.

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